Zeiss LSM 880 Operating Manual page 349

LSM 880
Feature size sets the mask size that is used to cut out the bead and to create the Experimental PSF.
The extensions are displayed in the Preview tab of the image container as circles (Fig. 473).
White circles indicate that the particular PSFs are used (if channel color is grey; otherwise the channel
color is used), red circles that the PSF are discarded for the calculation of the Experimental PSF.
Fig. 473
Noise Level sets the threshold of the intensity of the sub-resolution beads over background according to
I – M > SD x NL (I = intensity of bead; M = mean intensity of image frame; SD = standard deviation of
image frame; NL = noise level). Lowest number means that everything (even noise) is taken into account;
highest number means only the brightest beads will be used.
Beads quality evaluates two characteristics, the intensity (first moment) and the size (second moment).
These two moments are calculated for every localized bead. Then the beads are sorted according to these
characteristics, resulting in two sorted lists of beads. The beads that are closer to the medians in both
orderings are assumed to have higher quality. The Beads quality defines how far the beads
characteristics (intensity and size) may be away from the median value. The smaller the threshold, the
closer beads should be to the median. Beads quality equals to 0 is likely to result in a single bead right in
the middle. In this way also the bead shape (aspect ratio) is assessed. Lower input number means that
only perfectly shaped round beads will be taken into account. Highest input numbers will take into
account all beads, even ellipsoid shaped ones.
Focal Plane determines which plane is the focal plane. The default is -1 (automatic). Here the system
determines the focal plane automatically. The user can also select a frame, which is presumably the focal
plane, by input the number of the frame within the stack.
All sub-resolution beads that will be taken into account will be circled by a white line. Those that do
overlap with their circles or are considered unsuitable (beads beyond the quality thresholds) are outlined
in red and will not be taken into account (Fig. 473).
Please note that you should use a 4 µm stack of 100 nm beads, if you want to use the
Experimental PSF for 3D PALM.
10/2014 V_01
CHAPTER 1 - SYSTEM OPERATION
Left Tool Area and Hardware Control Tools
Selection of beads in Preview tab
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ZEISS
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